Protein biopolymer markers predictive of type II diabetes

ABSTRACT

The instant invention involves the use of a combination of preparatory steps in conjunction with mass spectroscopy and time-of-flight detection procedures to maximize the diversity of biopolymers which are verifiable within a particular sample. The cohort of biopolymers verified within such a sample is then viewed with reference to their ability to evidence at least one particular disease state; thereby enabling a diagnostician to gain the ability to characterize either the presence or absence of said at least one disease state relative to recognition of the presence and/or the absence of said biopolymer, predict disease risk assessment, and develop therapeutic avenues against said disease.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.09/993,393, filed on Nov. 23, 2001, the contents of which is hereinincorporated by reference.

FIELD OF THE INVENTION

This invention relates to the field of characterizing the existence of adisease state; particularly to the utilization of mass spectrometry toelucidate particular biopolymer markers indicative or predictive of aparticular disease state, and most particularly to specific biopolymermarkers whose up-regulation, down-regulation, or relative presence indisease vs. normal states has been determined to be useful in diseasestate assessment and therapeutic target recognition, development andvalidation.

BACKGROUND OF THE INVENTION

Methods utilizing mass spectrometry for the analysis of a targetpolypeptide have been taught wherein the polypeptide is firstsolubilized in an appropriate solution or reagent system. The type ofsolution or reagent system, e.g., comprising an organic or inorganicsolvent, will depend on the properties of the polypeptide and the typeof mass spectrometry performed and are well-known in the art (see, e.g.,Vorm et al. (1994) Anal. Chem. 66:3281 (for MALDI) and Valaskovic et al.(1995) Anal. Chem. 67:3802 (for ESI). Mass spectrometry of peptides isfurther disclosed, e.g., in WO 93/24834 by Chait et al.

In one prior art embodiment, the solvent is chosen so that the risk thatthe molecules may be decomposed by the energy introduced for thevaporization process is considerably reduced, or even fully excluded.This can be achieved by embedding the sample in a matrix, which can bean organic compound, e.g., sugar, in particular pentose or hexose, butalso polysaccharides such as cellulose. These compounds are decomposedthermolytically into CO₂ and H₂O so that no residues are formed whichmight lead to chemical reactions. The matrix can also be an inorganiccompound, e.g., nitrate of ammonium which is decomposed practicallywithout leaving any residues. Use of these and other solvents arefurther disclosed in U.S. Pat. No. 5,062,935 by Schlag et al.

Prior art mass spectrometer formats for use in analyzing the translationproducts include ionization (I) techniques, including but not limited tomatrix assisted laser desorption (MALDI), continuous or pulsedelectrospray (ESI) and related methods (e.g., IONSPRAY or THERMOSPRAY),or massive cluster impact (MCI); these ion sources can be matched withdetection formats including linear or non-linear reflectiontime-of-flight (TOF), single or multiple quadropole, single or multiplemagnetic sector, Fourier Transform ion cyclotron resonance (FTICR), iontrap, and combinations thereof (e.g., ion-trap/time-of-flight). Forionization, numerous matrix/wavelength combinations (MALDI) or solventcombinations (ESI) can be employed. Subattomole levels of protein havebeen detected, for example, using ESI (Valaskovic, G. A. et al., (1996)Science 273:1199-1202) or MALDI (Li, L. et al., (1996) J. Am. Chem. Soc.118:1662-1663) mass spectrometry.

ES mass spectrometry has been introduced by Fenn et al. (J. Phys. Chem.88, 4451-59 (1984); PCT Application No. WO 90/14148) and currentapplications are summarized in recent review articles (R. D. Smith etal., Anal. Chem. 62, 882-89 (1990) and B. Ardrey, Electrospray MassSpectrometry, Spectroscopy Europe, 4, 10-18 (1992)). MALDI-TOF massspectrometry has been introduced by Hillenkamp et al. (“Matrix AssistedUV-Laser Desorption/Ionization: A New Approach to Mass Spectrometry ofLarge Biomolecules,” Biological Mass Spectrometry (Burlingame andMcCloskey, editors), Elsevier Science Publishers, Amsterdam, pp. 49-60,1990). With ESI, the determination of molecular weights in femtomoleamounts of sample is very accurate due to the presence of multiple ionpeaks which all could be used for the mass calculation.

The mass of the target polypeptide determined by mass spectrometry isthen compared to the mass of a reference polypeptide of known identity.In one embodiment, the target polypeptide is a polypeptide containing anumber of repeated amino acids directly correlated to the number oftrinucleotide repeats transcribed/translated from DNA; from its massalone the number of repeated trinucleotide repeats in the original DNAwhich coded it, may be deduced.

U.S. Patent No. 6,020,208 utilizes a general category of probe elements(i.e., sample presenting means) with Surfaces Enhanced for LaserDesorption/Ionization (SELDI), within which there are three (3) separatesubcategories. The SELDI process is directed toward a sample presentingmeans (i.e., probe element surface) with surface-associated (orsurface-bound) molecules to promote the attachment (tethering oranchoring) and subsequent detachment of tethered analyte molecules in alight-dependent manner, wherein the said surface molecule(s) areselected from the group consisting of photoactive (photolabile)molecules that participate in the binding (docking, tethering, orcrosslinking) of the analyte molecules to the sample presenting means(by covalent attachment mechanisms or otherwise).

PCT/EP/04396 teaches a process for determining the status of an organismby peptide measurement. The reference teaches the measurement ofpeptides in a sample of the organism which contains both high and lowmolecular weight peptides and acts as an indicator of the organism'sstatus. The reference concentrates on the measurement of low molecularweight peptides, i.e. below 30,000 Daltons, whose distribution serves asa representative cross-section of defined controls. Contrary to themethodology of the instant invention, the '396 patent strives todetermine the status of a healthy organism, i.e. a “normal” and then usethis as a reference to differentiate disease states. The presentinventors do not attempt to develop a reference “normal”, but ratherstrive to specify particular markers whose presence, absence or relativestrength/concentration in disease vs. normal is diagnostic of at leastone specific disease state or whose up-regulation or down-regulation ispredictive of at least one specific disease state, whereby the presenceof said marker serves as a positive indicator useful in distinguishingdisease state. This leads to a simple method of analysis which caneasily be performed by an untrained individual, since there is apositive correlation of data. On the contrary, the '396 patent requiresa complicated analysis by a highly trained individual to determinedisease state versus the perception of non-disease or normal physiology.

Richter et al, Journal of Chromatography B, 726(1999) 25-35, refer to adatabase established from human hemofiltrate comprised of a massdatabase and a sequence database. The goal of Richter et al was toanalyze the composition of the peptide fraction in human blood. UsingMALDI-TOF, over 20,000 molecular masses were detected representing anestimated 5,000 different peptides. The conclusion of the study was thatthe hemofiltrate (HF) represented the peptide composition of plasma. Nocorrelation of peptides with relation to normal and/or disease states ismade.

As used herein, “analyte” refers to any atom and/or molecule; includingtheir complexes and fragment ions. The term may refer to a singlecomponent or a set of components. In the case of biologicalmolecules/macromolecules or “biopolymers”, such analytes include but arenot limited to: polypeptides, polynucleotides, proteins, peptides,antibodies, DNA, RNA, carbohydrates, steroids, and lipids, and anydetectable moiety thereof, e.g. immunologically detectable fragments.Note that most important biomolecules under investigation for theirinvolvement in the structure or regulation of life processes are quitelarge (typically several thousand times larger than H₂O).

As used herein, the term “molecular ions” refers to molecules in thecharged or ionized state, typically by the addition or loss of one ormore protons (H⁺).

As used herein, the term “molecular fragmentation” or “fragment ions”refers to breakdown products of analyte molecules caused, for example,during laser-induced desorption (especially in the absence of addedmatrix).

As used herein, the term “solid phase” refers to the condition of beingin the solid state, for example, on the probe element surface.

As used herein, “gas” or “vapor phase” refers to molecules in thegaseous state (i.e., in vacuo for mass spectrometry).

As used herein, the term “analyte desorption/ionization” refers to thetransition of analytes from the solid phase to the gas phase as ions.Note that the successful desorption/ionization of large, intactmolecular ions by laser desorption is relatively recent (circa 1988)—thebig breakthrough was the chance discovery of an appropriate matrix(nicotinic acid).

As used herein, the term “gas phase molecular ions” refers to those ionsthat enter into the gas phase. Note that large molecular mass ions suchas proteins (typical mass=60,000 to 70,000 times the mass of a singleproton) are typically not volatile (i.e., they do not normally enterinto the gas or vapor phase). However, in the procedure of the presentinvention, large molecular mass ions such as proteins do enter the gasor vapor phase.

As used herein in the case of MALDI, the term “matrix” refers to any oneof several small, acidic, light absorbing chemicals (e.g., CHCA(alpha-cyano-4-hydroxy-cinnamic acid), nicotinic or sinapinic acid) thatis mixed in solution with the analyte in such a manner so that, upondrying on the probe element, the crystalline matrix-embedded analytemolecules are successfully desorbed (by laser irradiation) and ionizedfrom the solid phase (crystals) into the gaseous or vapor phase andaccelerated as intact molecular ions. For the MALDI process to besuccessful, analyte is mixed with a freshly prepared solution of thechemical matrix (e.g., 10,000:1 matrix:analyte) and placed on the inertprobe element surface to air dry just before the mass spectrometricanalysis. The large fold molar excess of matrix, present atconcentrations near saturation, facilitates crystal formation andentrapment of analyte.

As used herein, “energy absorbing molecules (EAM)” refers to any one ofseveral small, light absorbing chemicals that, when presented on thesurface of a probe, facilitate the neat desorption of molecules from thesolid phase (i.e., surface) into the gaseous or vapor phase forsubsequent acceleration as intact molecular ions. The term EAM ispreferred, especially in reference to SELDI. Note that analytedesorption by the SELDI process is defined as a surface-dependentprocess (i.e., neat analyte may be placed on a surface composed of boundEAM or EAM and analyte may be mixed prior to placement on a surface). Incontrast, MALDI is presently thought to facilitate analyte desorption bya volcanic eruption-type process that “throws” the entire surface intothe gas phase. Furthermore, note that some EAM when used as freechemicals to embed analyte molecules as described for the MALDI processwill not work (i.e., they do not promote molecular desorption, thus theyare not suitable matrix molecules).

As used herein, “probe element” or “sample presenting device” refers toan element having the following properties: it is inert (for example,typically stainless steel) and active (probe elements with surfacesenhanced to contain EAM and/or molecular capture devices).

As used herein, “MALDI” refers to Matrix-Assisted LaserDesorption/Ionization.

As used herein, “TOF” stands for Time-of-Flight.

As used herein, “MS” refers to Mass Spectrometry.

As used herein, “MS/MS” refers to multiple sequential mass spectrometry.

As used herein “MALDI-TOF MS” refers to Matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry.

As used herein, “ESI” is an abbreviation for electrospray ionization.

As used herein, “chemical bonds” is used simply as an attempt todistinguish a rational, deliberate, and knowledgeable manipulation ofknown classes of chemical interactions from the poorly defined kind ofgeneral adherence observed when one chemical substance (e.g., matrix) isplaced on another substance (e.g., an inert probe element surface).Types of defined chemical bonds include electrostatic or ionic (+/−)bonds (e.g., between a positively and negatively charged groups on aprotein surface), covalent bonds (very strong or “permanent” bondsresulting from true electron sharing), coordinate covalent bonds (e.g.,between electron donor groups in proteins and transition metal ions suchas copper or iron), and hydrophobic interactions (such as between twononcharged groups), weak dipole and London force or induced dipoleinteractions.

As used herein, “electron donor groups” refers to the case ofbiochemistry, where atoms in biomolecules (e.g, N, S, O) “donate” orshare electrons with electron poor groups (e.g., Cu ions and othertransition metal ions).

As used herein, the term “biopolymer markers indicative or predictive ofa disease state” is interpreted to mean that a biopolymer marker whichis strongly present in a normal individual, but is down-regulated indisease is predictive of said disease; while alternatively, a biopolymermarker which is strongly present in a disease state, but isdown-regulated in normal individuals, is indicative of said diseasestate. Biopolymer markers which are present in both disease and normalstates are indicative/predictive based upon their relative strengths indisease vs. normal, along with the observation regarding when theirsignal strengthens/weakens relative to disease manifestation orprogression.

As used herein, the term “disease state assessment” is interpreted tomean quantitative or qualitative determination of the presence/absenceof the disease, with or without an ability to determine severity,rapidity of onset, or resolution of the disease state, e.g. a return toa normal physiological state.

As used herein, the term “therapeutic target recognition, development,and validation” refers to any concept or method which enables an artisanto recognize, develop, or validate the efficacy of a therapeutic moietywhich is effected in conjunction with a chemical or physical interactionwith one or more of the biopolymer markers of the instant invention.

As used herein, the term “polypeptide” is interpreted to mean a polymercomposed of amino acid residues, related naturally occurring structuralvariants, and synthetic non-naturally occurring analogs thereof linkedvia peptide bonds, related naturally occurring structural variants, andsynthetic non-naturally occurring analogs thereof. Syntheticpolypeptides can be synthesized, for example, using an automatedpolypeptide synthesizer. The term “protein” typically refers to largepolypeptides. The term “peptide” typically refers to short polypeptides.“Polypeptide(s)” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. “Polypeptide(s)” refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. “Polypeptide(s)” includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well-known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

As used herein, the term “polynucleotide” is interpreted to mean apolymer composed of nucleotide units. Polynucleotides include naturallyoccurring nucleic acids, such as deoxyribonucleic acid (“DNA”) andribonucleic acid (“RNA”) as well as nucleic acid analogs. Nucleic acidanalogs include those which include non-naturally occurring bases,nucleotides that engage in linkages with other nucleotides other thanthe naturally occurring phosphodiester bond or which include basesattached through linkages other than phosphodiester bonds. Thus,nucleotide analogs include, for example and without limitation,phosphorothioates, phosphorodithioates, phosphorotriesters,phosphoramidates, boranophosphates, methylphosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs),and the like. Such polynucleotides can be synthesized, for example,using an automated DNA synthesizer. The term “nucleic acid” typicallyrefers to large polynucleotides. The term “oligonucleotide” typicallyrefers to short polynucleotides, generally no greater than about 50nucleotides. It will be understood that when a nucleotide sequence isrepresented by a DNA sequence (i.e., A, T, G, C), this also includes anRNA sequence (i.e., A, U, G, C) in which “U” replaces T.

As used herein, the term “detectable moiety” or a “label” refers to acomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, or chemical means. For example, useful labels include³²P, ³⁵S, fluorescent dyes, electron-dense reagents, enzymes (e.g., ascommonly used in an ELISA), biotin-streptavadin, dioxigenin, haptens andproteins for which antisera or monoclonal antibodies are available, ornucleic acid molecules with a sequence complementary to a target. Thedetectable moiety often generates a measurable signal, such as aradioactive, chromogenic, or fluorescent signal, that can be used toquantitate the amount of bound detectable moiety in a sample. Thedetectable moiety can be incorporated in or attached to a primer orprobe either covalently, or through ionic, van der Waals or hydrogenbonds, e.g., incorporation of radioactive nucleotides, or biotinylatednucleotides that are recognized by streptavadin. The detectable moietymay be directly or indirectly detectable. Indirect detection can involvethe binding of a second directly or indirectly detectable moiety to thedetectable moiety. For example, the detectable moiety can be the ligandof a binding partner, such as biotin, which is a binding partner forstreptavadin, or a nucleotide sequence, which is the binding partner fora complementary sequence, to which it can specifically hybridize. Thebinding partner may itself be directly detectable, for example, anantibody may be itself labeled with a fluorescent molecule. The bindingpartner also may be indirectly detectable, for example, a nucleic acidhaving a complementary nucleotide sequence can be a part of a branchedDNA molecule that is in turn detectable through hybridization with otherlabeled nucleic acid molecules. (See, e.g., P. D. Fahrlander and A.Klausner, Bio/Technology (1988) 6:1165.) Quantitation of the signal isachieved by, e.g., scintillation counting, densitometry, or flowcytometry.

As used herein, the term “antibody or antibodies”includes polyclonal andmonoclonal antibodies of any isotype (IgA, IgG, IgE, IgD, IgM), or anantigen-binding portion thereof, including but not limited to F(ab) andFv fragments, single chain antibodies, chimeric antibodies, humanizedantibodies, and a Fab expression library. “Antibody” refers to apolypeptide ligand substantially encoded by an immunoglobulin gene orimmunoglobulin genes, or fragments thereof, which specifically binds andrecognizes an epitope (e.g., an antigen). The recognizedimmunoglobulin—genes include the kappa and lambda light chain constantregion genes, the alpha, gamma, delta, epsilon and mu heavy chainconstant region genes, and the myriad immunoglobulin variable regiongenes. Antibodies exist, e.g., as intact immunoglobulins or as a numberof well characterized fragments produced by digestion with variouspeptidases. This includes, e.g., Fab′ and F(ab)′₂ fragments. The term“antibody,” as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA methodologies. It also includes polyclonalantibodies, monoclonal antibodies, chimeric antibodies and humanizedantibodies. “Fc” portion of an antibody refers to that portion of animmunoglobulin heavy chain that comprises one or more heavy chainconstant region domains, CH, CH₂ and CH₃, but does not include the heavychain variable region.

As used herein, the term “moieties” refers to an indefinite portion of asample.

A “ligand” is a compound that specifically binds to a target molecule.

A “receptor” is a compound or portion of a structure that specificallybinds to a ligand.

A ligand or a receptor (e.g., an antibody) “specifically binds to” or“is specifically immunoreactive with” a compound analyte when the ligandor receptor functions in a binding reaction which is determinative ofthe presence of the analyte in a sample of heterogeneous compounds.Thus, under designated assay (e.g., immunoassay) conditions, the ligandor receptor binds preferentially to a particular analyte and does notbind in a significant amount to other compounds present in the sample.For example, a polynucleotide specifically binds under hybridizationconditions to an analyte polynucleotide comprising a complementarysequence; an antibody specifically binds under immunoassay conditions toan antigen analyte bearing an epitope against which the antibody wasraised; and an adsorbent specifically binds to an analyte under properelution conditions.

As used herein, the term “pharmaceutically effective carrier” refers toany solid or liquid material which may be used in creating formulationsthat further include active ingredients of the instant invention, e.g.biopolymer markers or therapeutics, for administration to a patient.

As used herein, the term “agent” is interpreted to mean a chemicalcompound, a mixture of chemical compounds, a sample of undeterminedcomposition, a combinatorial small molecule array, a biologicalmacromolecule, a bacteriophage peptide display library, a bacteriophageantibody (e.g., scFv) display library, a polysome peptide displaylibrary, or an extract made from biological materials such as bacteria,plants, fungi, or animal cells or tissues. Suitable techniques involveselection of libraries of recombinant antibodies in phage or similarvectors. See, Huse et al. (1989) Science 246: 1275-1281; and Ward et al.(1989) Nature 341: 544-546. The protocol described by Huse is renderedmore efficient in combination with phage display technology. See, e.g.,Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047.

As used herein, the term “isolated” is interpreted to mean altered “bythe hand of man” from its natural state, i.e., if it occurs in nature,it has been changed or removed from its original environment, or both.For example, a polynucleotide or a polypeptide naturally present in aliving organism is not “isolated,” but the same polynucleotide orpolypeptide separated from the coexisting materials of its natural stateis “isolated”, as the term is employed herein.

As used herein, the term “variant” is interpreted to mean apolynucleotide or polypeptide that differs from a referencepolynucleotide or polypeptide respectively, but retains essentialproperties. A typical variant of a polynucleotide differs in nucleotidesequence from another, reference polynucleotide. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. A variant ofa polynucleotide or polypeptide may be a naturally occurring such as anallelic variant, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques, by direct synthesis,and by other recombinant methods known to skilled artisans.

As used herein, the term “biopolymer marker” refers to a polymer ofbiological origin, e.g. polypeptides, polynucleotides, polysaccharidesor polyglycerides (e.g., di- or tri-glycerides), and may include anyfragment, e.g. immunologically reactive fragments, variants or moietiesthereof.

As used herein, the term “fragment” refers to the products of thechemical, enzymatic, or physical breakdown of an analyte. Fragments maybe in a neutral or ionic state.

As used herein, the term “therapeutic avenues” is interpreted to meanany agents, modalities, synthesized compounds, etc., which interact witha biopolymer marker in any manner that facilitates a therapeuticbenefit, including immunotherapeutic intervention, e.g. modalities suchas administration of an immunologically reactive moiety capable ofaltering the course, progression and/or manifestation of the disease, asa result of interfering with the disease manifestation process, forexample, at the early stages focused upon by the identification of thedisease, such as by supplying a moiety capable of modifying thepathogenicity of lymphocytes specific for the biopolymer marker orrelated components.

As used herein, the term “interacting with a biopolymer marker” includesany process by which a biopolymer marker may physically or chemicallyrelate with an organism, particularly when this interaction results inthe development of therapeutic avenues or in modulation of the diseasestate.

As used herein, the term “therapeutic targets” may thus be defined asthose analytes which are capable of exerting a modulating force, wherein“modulation” is defined as an alteration in function inclusive ofactivity, synthesis, production, and circulating levels. Thus,modulation effects the level or physiological activity of at least oneparticular disease related biopolymer marker or any compound orbiomolecule whose presence, level or activity is linked either directlyor indirectly, to an alteration of the presence, level, activity orgeneric function of the biopolymer marker, and may includepharmaceutical agents, biomolecules that bind to the biopolymer markers,or biomolecules or complexes to which the biopolymer markers bind. Thebinding of the biopolymer markers and the therapeutic moiety may resultin activation (agonist), inhibition (antagonist), or an increase ordecrease in activity or production (modulator) of the biopolymer markersor the bound moiety. Examples of such therapeutic moieties include, butare not limited to, antibodies, oligonucleotides, proteins (e.g.,receptors), RNA, DNA, enzymes, peptides or small molecules. With regardto immunotherapeutic moieties, such a moiety may be defined as aneffective analog for a major epitope peptide which has the ability toreduce the pathogenicity of key lymphocytes which are specific for thenative epitope. An analog is defined as having structural similarity butnot identity in peptide sequencing able to be recognized by T-cellsspontaneously arising and targeting the endogeneous self epitope. Acritical function of this analog is an altered T-cell activation whichleads to T-cell anergy or death.

With the advent of mass spectrometric methods such as MALDI and SELDIand ESI, researchers have begun to utilize a tool that holds the promiseof uncovering countless biopolymers which result from translation,transcription and post-translational transcription of proteins from theentire genome.

Operating upon the principles of retentate chromatography, SELDI MSinvolves the adsorption of proteins, based upon their physico-chemicalproperties at a given pH and salt concentration, followed by selectivelydesorbing proteins from the surface by varying pH, salt, or organicsolvent concentration. After selective desorption, the proteins retainedon the SELDI surface, the “chip”, can be analyzed using the CIPHERGENprotein detection system, or an equivalent thereof. Retentatechromatography is limited, however, by the fact that if unfractionatedbody fluids, e.g. blood, blood products, urine, saliva, cerebrospinalfluid, luymph and the like, along with tissue samples, are applied tothe adsorbent surfaces, the biopolymers present in the greatestabundance will compete for all the available binding sites and therebyprevent or preclude less abundant biopolymers from interacting withthem, thereby reducing or eliminating the diversity of biopolymers whichare readily ascertainable.

If a process could be devised for maximizing the diversity ofbiopolymers discernable from a sample, the ability of researchers toaccurately determine the relevance of such biopolymers with relation toone or more disease states would be immeasurably enhanced.

SUMMARY OF THE INVENTION

The instant invention is characterized by the use of a combination ofpreparatory steps, e.g. chromatography and 1-D tricine polyacrylamidegel electrophoresis. Subsequent to which the gel is stained, e.g. withCoomasie blue, silver or rubidium. Next, bands are selected from thegels for further study. Tryptic digestion of each band follows,concluding with the extraction of tryptic peptides from the digest. Thisextraction may be accomplished utilizing C18 ZIPTIPs, or organic extractand dry technique followed by MALDI Qq TOF (Maldi Quadrupole QuadrupoleTime of Flight) processing.

Additional methodologies may include SELDI MS, 2-D gel technology, MALDIMS/MS and time-of-flight detection procedures to maximize the diversityof biopolymers which are verifiable within a particular sample. Thecohort of biopolymers verified within a sample is then compared todevelop data indicating their presence, absence or relativestrength/concentration in disease vs normal controls, and furtherstudied to determine whether the up-regulation or down-regulation of asingle biopolymer or group of biopolymers is indicative of a diseasestate or predictive of the development of said disease state.Additionally, biopolymers recognized as being indicative or predictiveof a disease state in accordance with the instant invention are usefulin therapeutic intervention, e.g. as therapeutic modalities in their ownright, in the course of therapeutic target recognition, in thedevelopment and validation of efficacious therapeutic modalities, e.gwhen interrogating or developing phage display libraries, and as ligandsor receptors for use in conjunction with therapeutic intervention.

Although all manner of biomarkers related to all disease conditions aredeemed to be within the purview of the instant invention andmethodology, particular significance was given to those markers anddiseases associated with the complement system, cognitive diseases, e.g.Alzheimer's disease and Syndrome X and diseases related thereto.

The complement system is an important part of non-clonal or innateimmunity that collaborates with acquired immunity to destroy invadingpathogens and to facilitate the clearance of immune complexes from thesystem. This system is the major effector of the humoral branch of theimmune system, consisting of nearly 30 serum and membrane proteins. Theproteins and glycoproteins composing the complement system aresynthesized largely by liver hepatocytes. Activation of the complementsystem involves a sequential enzyme cascade in which the proenzymeproduct of one step becomes the enzyme catalyst of the next step.Complement activation can occur via two pathways: the classical and thealternative. The classical pathway is commonly initiated by theformation of soluble antigen-antibody complexes or by the binding ofantibody to antigen on a suitable target, such as a bacterial cell. Thealternative pathway is generally initiated by various cell-surfaceconstituents that are foreign to the host. Each complement component isdesignated by numerals (C1-C9), by letter symbols, or by trivial names.After a component is activated, the peptide fragments are denoted bysmall letters. The complement fragments interact with one another toform functional complexes. Ultimately, foreign cells are destroyedthrough the process of a membrane-attack complex mediated lysis.

The C4 component of the complement system is involved in the classicalactivation pathway. It is a glycoprotein containing three polypeptidechains (α, β, and γ). C4 is a substrate of component C1s and isactivated when C1s hydrolyzes a small fragment (C4a) from the aminoterminus of the α chain, exposing a binding site on the larger fragment(C4b).

The native C3 component consists of two polypeptide chains, α and β. Asa serum protein, C3 is involved in the alternative pathway. Serum C3,which contains an unstable thioester bond, is subject to slowspontaneous hydrolysis into C3a and C3b. The C3f component is involvedin the regulation required of the complement system which confines thereaction to designated targets. During the regulation process, C3b iscleaved into two parts: C3bi and C3f. C3bi is a membrane-boundintermediate wherein C3f is a free diffusible (soluble) component.

Complement components have been implicated in the pathogenesis ofseveral disease conditions. C3 deficiencies have the most severeclinical manifestations, such as recurrent bacterial infections andimmune-complex diseases, reflecting the central role of C3. The rapidprofusion of C3f moieties and resultant “accidental” lysis of normalcells mediated thereby gives rise to a host of auto-immune reactions.The ability to understand and control these mechanisms, along with theirattendant consequences, will enable practitioners to develop bothdiagnostic and therapeutic avenues by which to thwart these maladies.

In the course of defining a plurality of disease specific markersequences, special significance was given to markers which wereevidentiary of a particular disease state or with conditions associatedwith Syndrome-X. Syndrome-X is a multifaceted syndrome, which occursfrequently in the general population. A large segment of the adultpopulation of industrialized countries develops this metabolic syndrome,produced by genetic, hormonal and lifestyle factors such as obesity,physical inactivity and certain nutrient excesses. This disease ischaracterized by the clustering of insulin resistance andhyperinsulinemia, and is often associated with dyslipidemia (atherogenicplasma lipid profile), essential hypertension, abdominal (visceral)obesity, glucose intolerance or noninsulin-dependent diabetes mellitusand an increased risk of cardiovascular events. Abnormalities of bloodcoagulation (higher plasminogen activator inhibitor type I andfibrinogen levels), hyperuricemia and microalbuminuria have also beenfound in metabolic syndrome-X.

The instant inventors view the Syndrome X continuum in itscardiovascular light, while acknowledging its important metaboliccomponent. The first stage of Syndrome X consists of insulin resistance,abnormal blood lipids (cholesterol, triglycerides and free fatty acids),obesity, and high blood pressure (hypertension). Any one of these fourfirst stage conditions signals the start of Syndrome X.

Each first stage Syndrome X condition risks leading to another. Forexample, increased insulin production is associated with high blood fatlevels, high blood pressure, and obesity. Furthermore, the effects ofthe first stage conditions are additive; an increase in the number ofconditions causes an increase in the risk of developing more seriousdiseases on the Syndrome X continuum.

A patient who begins the Syndrome X continuum risks spiraling into amaze of increasingly deadly diseases. The next stages of the Syndrome Xcontinuum lead to overt diabetes, kidney failure, and heart failure,with the possibility of stroke and heart attack at any time. Syndrome Xis a dangerous continuum, and preventative medicine is the best defense.Diseases are currently most easily diagnosed in their later stages, butcontrolling them at a late stage is extremely difficult. Diseaseprevention is much more effective at an earlier stage.

In a further contemplated embodiment of the invention, samples may betaken from a patient at one point in time, as a single sample or asmultiple samples, or at different points in time such that analysis iscarried out on multiple samples for ongoing analysis. Typically, a firstsample is taken from a patient upon presentation with possible symptomsof a disease and analyzed according to the invention. Subsequently, someperiod of time after presentation, for example, about 3-6 months afterthe first presentation, a second sample is taken and analyzed accordingto the invention. The data can be used, by way of example, to diagnoseor monitor a disease state, determine risk assessment, identifytherapeutic avenues, or determine the therapeutic value of an agent suchas a pharmaceutical.

Subsequent to the isolation of particular disease state marker sequencesas taught by the instant invention, the promulgation of various forms ofrisk assessment tests are contemplated which will allow physicians toidentify asymptomatic patients before they suffer an irreversible eventsuch as diabetes, kidney failure, and heart failure, and enableeffective disease management and preventative medicine. Additionally,the specific diagnostic tests which evolve from this methodology providea tool for rapidly and accurately diagnosing acute Syndrome X eventssuch as heart attack and stroke, and facilitate treatment.

More particularly, biopolymer markers elucidated via methodologies ofthe instant invention find utility related to broad areas of diseasetherapeutics. Such therapeutic avenues include, but are not limited to:

-   1) utilization and recognition of said biopolymer markers, variants    or moieties thereof as direct therapeutic modalities, either alone    or in conjunction with an effective amount of a pharmaceutically    effective carrier;-   2) validation of therapeutic modalities or disease preventative    agents as a function of biopolymer marker presence or concentration;-   3) treatment or prevention of a disease state by formation of    disease intervention modalities; e.g. formation of biopolymer/ligand    conjugates which intervene at receptor sites to prevent, delay or    reverse a disease process;-   4) use of biopolymer markers or moieties thereof as a means of    elucidating therapeutically viable agents, e.g. from a bacteriophage    peptide display library, a bacteriophage antibody library or the    like;-   5) instigation of a therapeutic immunological response; and-   6) synthesis of molecular structures related to said biopolymer    markers, moieties or variants thereof which are constructed and    arranged to therapeutically intervene in the disease process.

A process for identifying or developing therapeutic avenues related to adisease state utilizing any of the above examples may follow resultsobtained from conducting an analysis inclusive of interacting with abiopolymer including the sequence of the particular disease specificmarker or at least one analyte thereof of the present invention. Suchtreatment or prevention of a disease state by formation of diseaseintervention modalities may be by the formation of biopolymer/ligandconjugates which intervene at receptor sites to prevent, delay, orreverse a disease process. In addition, a means of elucidatingtherapeutically viable agents may include the use of a bacteriophagepeptide display library or a bacteriophage antibody library. Thetherapeutic avenues may regulate the presence or absence of thebiopolymer including the sequence of the particular disease specificmarker or at least one analyte thereof in the present invention.

Accordingly, it is an objective of the instant invention to define adisease specific biopolymer marker sequence which is useful inevidencing and categorizing at least one particular disease state.

It is an additional objective of the instant invention to developmethods and means of disease therapy, including but not limited to:

-   1) utilization and recognition of said biopolymer markers, variants    or moieties thereof as direct therapeutic modalities, either alone    or in conjunction with an effective amount of a pharmaceutically    effective carrier;-   2) validation of therapeutic modalities or disease preventative    agents as a function of biopolymer marker presence or concentration;-   3) treatment or prevention of a disease state by formation of    disease intervention modalities; e.g. formation of biopolymer/ligand    conjugates which intervene at receptor sites to prevent, delay or    reverse a disease process;-   4) use of biopolymer markers or moieties thereof as a means of    elucidating therapeutically viable agents, e.g. from a bacteriophage    peptide display library, a bacteriophage antibody library or the    like;-   5) instigation of a therapeutic immunological response; and-   6) synthesis of molecular structures related to said biopolymer    markers, moieties or variants thereof which are constructed and    arranged to therapeutically intervene in the disease process, e.g.    by directly determining the three-dimensional structure of said    biopolymer marker directly from an amino acid sequence thereof.

It is another objective of the instant invention to evaluate samplescontaining a plurality of biopolymers for the presence of diseasespecific biopolymer marker sequences (disease specific markers) whichevidence a link to at least one specific disease state.

It is a further objective of the instant invention to elucidateessentially all biopolymeric markers, moieties or variants thereofcontained within said samples, whereby particularly significant moietiesmay be identified.

It is a further objective of the instant invention provide at least onepurified antibody which is specific to said disease specific markersequence.

It is yet another objective of the instant invention to teach amonoclonal antibody which is specific to said disease specific markersequence.

It is a still further objective of the invention to teach polyclonalantibodies raised against said disease specific marker.

It is yet an additional objective of the instant invention to teach adiagnostic kit for determining the presence, concentration, or relativestrength/concentration of said disease specific marker.

It is a still further objective of the instant invention to teachmethods for characterizing disease state based upon the identificationof said disease specific marker.

Other objects and advantages of this invention will become apparent fromthe following description taken in conjunction with the accompanyingdrawings wherein are set forth, by way of illustration and example,certain embodiments of this invention. The drawings constitute a part ofthis specification and include exemplary embodiments of the presentinvention and illustrate various objects and features thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a photograph of a tricine gel comparing a normal specimenversus Type II Diabetes;

FIG. 2 is a trypsin digested spectra graph depicting the ion 1198;

FIG. 3 is a trypsin digested spectra graph depicting the ion 1969; and

FIG. 4 is a trypsin digested spectra graph depicting the ion 1301.

DETAILED DESCRIPTION OF THE INVENTION

In earlier work, for example in U.S. patent application Ser. No.09/846330 filed Apr. 30, 2000, the contents of which is hereinincorporated by reference, raw sera was obtained and mixed with formicacid and extracted the peptides with C18 reversed phase ZIPTIPs.

In the instantly disclosed invention, we deal with proteins generallyhaving a molecular weight of about 20 kD or more. In general, proteinsof greater than 20 kD can reliably be fragmented by trypsin or otherenzymes. The instant technology incorporates sufficient sensitivity todeal with even the low production of peptides from proteins less than 20kD clipped from gel.

Proteins differ from peptides in that they cannot be effectivelyresolved by time of flight MS and they are too large (>3 kD) to beeffectively fragmented by collision with gases. The most commonly usedsolution to these problems is to resolve the proteins by polyacrylamidegel electrophoresis followed by staining with silver, or coomasiebrilliant blue or rubidium dyes or counter staining with Zinc-SDScomplexes. Once the proteins have been resolved and visualized withstains the proteins that differ between disease states can then beexcised from the gel and the protein purified in the 1-D gel band or 2-Dgel spot can be cleaved into fragments less that 3 kD by proteolyticenzymes. Once protein has been resolved by gel and cleaved by enzymes,the protein is considered in the form of peptides and therefore can bedealt with as per earlier work (09/846330). The peptide is eithercollected and purified with C18 reversed phase chromatography or by someother form of chromatography prior to reversed phase separation. Thepeptide can also be collected in ammonium carbonate buffer that issubsequently evolved by reaction with acid or by removal in organicsolvents.

Once the peptides are collected they can be sequenced, e.g. with aMALDI-Qq-TOF but also with a TOF-TOF, and ESI-Q-TOF or an ION-TRAP.Other types of MS analysis which may be employed are SELDI MS and MS/MS.The peptides are fragments of the original protein. The peptides aresequenced by fragmentation to produced a spectrum composed of the partsof the peptide. The peptide fragments can be produced by a strongionization energy with a laser, temperature, electron capture, collisionbetween the peptides themselves or with other objects such as gasmolecules. The spacing in terms of mass between the parts of thepeptides is a fragmentation pattern. The fragmentation pattern of eachpeptide from the starting mass to the last remaining amino acid (fromeither end) is unique.

The human genome contains the genes that encode all proteins. Theproteolytic cut sites within all these proteins can be predicted fromthe translated amino acid sequence. The mass of the peptides that resultfrom the predicting cut sites can be calculated. Similarly, thefragmentation pattern from each hypothetical peptide can be predicted.Thus, we can conceptually digest the proteins within the human proteomeand fragment them.

When a peptide has been “sequenced” it is understood that the peptidefragment has been purified by one of the methods above, i.e. Time offlight (TOF) or by chromatography, before fragmenting it with gas toproduce the peptide fragments. The original peptide mass andfragmentation pattern obtained is then fit to those from the theoreticaldigestion and fragmentation of the genome. The peptide that best matchesthe theoretical peptides and fragments and is biologically possible,i.e. a potential human blood-borne protein, is thus identified. It ispossible to identify plural targets in this fashion.

Following are exemplary, but non-limiting examples of preparatoryprotocols useful in the process of the instant invention.

Preparatory Protocols:

Any of these protocols may be selected from a column flow-throughstream, a column elution stream, or a column scrub stream.

-   Hi Q is a strong anion exchanger made of methyl acrylate co-polymer    with the functional group: —N⁺(CH₃)₂;-   Hi S is a strong cation exchanger made of methyl acrylate co-polymer    with the functional group: —SO₃ ⁻;-   DEAE is diethylaminoethyl which is a weak cation exchanger made of    methyl acrylate co-polymer with the functional group —N⁺(C₂H₅)₂;-   PS is phenyl sepharose;-   BS is butyl sepharose.    Note that the supports, i.e. methyl acrylate and sepharose are    different, but non-limiting examples, as the same functional group    on different supports will function, albeit possibly with different    effects.

DEAE Column Protocol:

-   1) Cast 200 μl of 50% slurry;-   2) Equilibrate column in 5 bed volumes of 50 mM tricine pH 8.8    (binding buffer);-   3) Dissolve 25 μl of sera in 475 μl of binding buffer;-   4) Wash column in 5 bed volumes of binding buffer;-   5) Elute column in 120 μl of 0.4 M Phosphate buffer (PB) pH 6.1;-   6) Elute column in 120 μl of 50 mM citrate buffer pH 4.2;-   7) Scrub column with 120 μl sequentially with each of 0.1% triton,    1.0% triton and 2% SDS in 62.5 mM Tris pH 6.8.

Butyl Sepharose Column Protocol:

-   1) Cast 150 μl bed volume column;-   2) Equilibrate column in 5 bed volumes of 1.7 M (NH₄)₂SO₄ in 50 mM    PB pH 7.0 (binding buffer);-   3) Dissolve 35 μl of sera in 465 μl of binding buffer and apply;-   4) Wash column in 5 bed volumes of binding buffer;-   5) Elute column in 120 μl of 0.4 M (NH₄)₂SO₄ in 50 mM PB pH 7.0;-   6) Elute column in 120 μl of 50 mM PB pH 7.0;-   7) Scrub column with 120 μl sequentially with each of 0.1% triton,    1.0% triton and 2% SDS in 62.5 mM Tris pH 6.8.

Phenyl Sepharose Column Protocol:

-   1) Cast 150 μl bed volume column;-   2) Equilibrate column in 5 bed volumes of 1.7 M (NH₄)₂SO₄ in 50 mM    PB pH 7.0 (binding buffer);-   3) Dissolve 35 μl of sera in 465 μl of binding buffer and apply;-   4) Wash column in 5 bed volumes of binding buffer;-   5) Elute column in 120 μl of 0.2 M (NH₄)₂SO₄ in 50 mM PB pH 7.0;-   6) Elute column in 120 μl of 50 mM PB pH 7.0;-   7) Scrub column with 120 μl sequentially with each of 0.1% triton,    1.0% triton and 2% SDS in 62.5 mM Tris pH 6.8.

HiQ Anion Exchange Mini Column Protocol:

-   1) Dilute sera in sample/running buffer;-   2) Add HiQ resin to column and remove any air bubbles;-   3) Add ultrafiltered (UF) water to aid in column packing;-   4) Add sample/running buffer to equilibrate column;-   5) Add diluted sera;-   6) Collect all the flow-through fraction in Eppendorf tubes until    level is at resin;-   7) Add sample/running buffer to wash column;-   8) Add elution buffer and collect elution in Eppendorf tubes.

HiS Cation Exchange Mini Column Protocol:

-   1) Dilute sera in sample/running buffer;-   2) Add HiS resin to column and remove any air bubbles;-   3) Add UF water to aid in column packing;-   4) Add sample/running buffer to equilibrate column for sample    loading;-   5) Add diluted sera to column;-   6) Collect all flow through fractions in Eppendorf tubes until level    is at resin;-   7) Add sample/running buffer to wash column;-   8) Add elution buffer and collect elution in Eppendorf tubes.

Illustrative of the various buffering compositions useful in thistechnique are: Sample/Running buffers: including but not limited toBicine buffers of various molarities, pH's, NaCl content, Bis-Trisbuffers of various molarities, pH's, NaCl content, Diethanolamine ofvarious molarities, pH's, NaCl content, Diethylamine of variousmolarities, pH's, NaCl content, Imidazole of various molarities, pH's,NaCl content, Tricine of various molarities, pH's, NaCl content,Triethanolamine of various molarities, pH's, NaCl content, Tris ofvarious molarities, pH's, NaCl content. Elution Buffer: Acetic acid ofvarious molarities, pH's, NaCl content, Citric acid of variousmolarities, pH's, NaCl content, HEPES of various molarities, pH's, NaClcontent, MES of various molarities, pH's, NaCl content, MOPS of variousmolarities, pH's, NaCl content, PIPES of various molarities, pH's, NaClcontent, Lactic acid of various molarities, pH's, NaCl content,Phosphate of various molarities, pH's, NaCl content, Tricine of variousmolarities, pH's, NaCl content.

Following tryptic digestion, additional processing may be carried out,for example:

Utilizing a type of micro-chromatographic column called a C18-ZIPTIPavailable from the Millipore company, the following preparatory stepswere conducted.

-   1. Dilute sera in sample buffer-   2. Aspirate and dispense ZIPTIP in 50% Acetonitrile-   3. Aspirate and dispense ZIPTIP in Equilibration solution-   4. Aspirate and dispense in serum sample-   5. Aspirate and dispense ZIPTIP in Wash solution-   6. Aspirate and dispense ZIPTIP in Elution Solution

Illustrative of the various buffering compositions useful in the presentinvention are: Sample Buffers (various low pH's): Hydrochloric acid(HCl), Formic acid, Trifluoroacetic acid (TFA), Equilibration Buffers(various low pH's): HCl, Formic acid, TFA;

Wash Buffers (various low pH's): HC, Formic acid, TFA;

Elution Solutions (various low pH's and % Solvents) HCl, Formic acid,TFA;

Solvents: Ethanol, Methanol, Acetonitrile.

Spotting was then performed, for example upon a Gold Chip in thefollowing manner:

-   1. Spot 2 ul of sample onto each spot-   2. Let sample partially dry

As a result of these procedures, the disease specific markers Actin betahaving a molecular weight of about 1198.7061 daltons and a sequence of(R)AVFPSIVGRPR(H), complement C3 precursor having a molecular weight ofabout 1969.9355 daltons and a sequence of (D)IPPADLSDQVPDTESETR(I),proapolipoprotein having a molecular weight of about 1301.6490 daltonsand a sequence of (R)THLAPYSDELR(Q), apolipoprotein A-IV precursor,having a molecular weight of about 1353 and sequence (R)RVEPYGENFNK(A)and a molecular weight of about 1104 and sequence (R)LEPYADQLR(T)predictive of Type II diabetes.

FIG. 1 is a photograph of a gel which is indicative of thepresence/absence of the marker in disease vs. control and, in caseswhere the marker is always present, the relative strength, e.g. the upor down regulation of the marker relative to categorization of diseasestate is deduced.

A method for evidencing and categorizing at least one disease state isdisclosed. The steps taken include obtaining a sample from a patient,preferably human, and conducting MS analysis on the sample. As a result,at least one biopolymer marker sequence or analyte thereof is isolatedfrom the sample which undergoes evidencing and categorizing and iscompared to the biopolymer marker sequence as disclosed in the presentinvention. The step of evidencing and categorizing is particularlydirected to biopolymer markers or analytes thereof linked to at leastone risk of disease development of the patient or related to theexistence of a particular disease state.

In addition, various kits are contemplated for use by the presentinvention. One such kit provides for determining the presence of thedisease specific biopolymer marker. At least one biochemical material isincorporated which is capable of specifically binding with a biomoleculewhich includes at least the disease specific biopolymer marker oranalyte thereof, and a means for determining binding between thebiochemical material and the biomolecule. The biochemical material forany of the contemplated kits, by way of example an antibody or at leastone monoclonal antibody specific therefore, or biomolecule may beimmobilized on a solid support and include at least one labeledbiochemical material which is preferably an antibody. The sampleutilized for any of the kits may be a fractionated or unfractionatedbody fluid or a tissue sample. Non-limiting examples of such fluids areblood, blood products, urine, saliva, cerebrospinal fluid, and lymph.

Further contemplated is a kit for diagnosing, determiningrisk-assessment, and identifying therapeutic avenues related to adisease state. This kit includes at least one biochemical material whichis capable of specifically binding with a biomolecule which includes atleast one biopolymer marker including the sequence of the particulardisease specific biopolymer marker or an analyte thereof related to thedisease state. Also included is a means for determining binding betweenthe biochemical material and the biomolecule, whereby at least oneanalysis to determine a presence of a marker, analyte thereof, or abiochemical material specific thereto, is carried out on a sample. Aspreviously described, analysis may be carried out on a single sample ormultiple samples.

In accordance with various stated objectives of the invention, theskilled artisan, in possession of the specific disease specific markeras instantly disclosed, would readily carry out known techniques inorder to raise purified biochemical materials, e.g. monoclonal and/orpolyclonal antibodies, which are useful in the production of methods anddevices useful as point-of-care rapid assay diagnostic or riskassessment devices as are known in the art.

The specific disease markers which are analyzed according to the methodof the invention are released into the circulation and may be present inthe blood or in any blood product, for example plasma, serum, cytolyzedblood, e.g. by treatment with hypotonic buffer or detergents anddilutions and preparations thereof, and other body fluids, e.g. CSF,saliva, urine, lymph, and the like. The presence of each marker isdetermined using antibodies specific for each of the markers anddetecting specific binding of each antibody to its respective marker.Any suitable direct or indirect assay method may be used to determinethe level of each of the specific markers measured according to theinvention. The assays may be competitive assays, sandwich assays, andthe label may be selected from the group of well-known labels such asradioimmunoassay, fluorescent or chemiluminescence immunoassay, orimmunoPCR technology. Extensive discussion of the known immunoassaytechniques is not required here since these are known to those ofskilled in the art. See Takahashi et al. (Clin Chem 1999;45(8):1307) fora detailed example of an assay.

A monoclonal antibody specific against the disease marker sequenceisolated by the present invention may be produced, for example, by thepolyethylene glycol (PEG) mediated cell fusion method, in a mannerwell-known in the art.

Traditionally, monoclonal antibodies have been made according tofundamental principles laid down by Kohler and Milstein. Mice areimmunized with antigens, with or without, adjuvants. The splenocytes areharvested from the spleen for fusion with immortalized hybridomapartners. These are seeded into microtiter plates where they can secreteantibodies into the supernatant that is used for cell culture. To selectfrom the hybridomas that have been plated for the ones that produceantibodies of interest, the hybridoma supernatants are usually testedfor antibody binding to antigens in an ELISA (enzyme linkedimmunosorbent assay) assay. The idea is that the wells that contain thehybridoma of interest will contain antibodies that will bind most avidlyto the test antigen, usually the immunizing antigen. These wells arethen subcloned in limiting dilution fashion to produce monoclonalhybridomas. The selection for the clones of interest is repeated usingan ELISA assay to test for antibody binding. Therefore, the principlethat has been propagated is that in the production of monoclonalantibodies the hybridomas that produce the most avidly bindingantibodies are the ones that are selected from among all the hybridomasthat were initially produced. That is to say, the preferred antibody isthe one with highest affinity for the antigen of interest.

There have been many modifications of this procedure such as using wholecells for immunization. In this method, instead of using purifiedantigens, entire cells are used for immunization. Another modificationis the use of cellular ELISA for screening. In this method instead ofusing purified antigens as the target in the ELISA, fixed cells areused. In addition to ELISA tests, complement mediated cytotoxicityassays have also been. used in the screening process. However,antibody-binding assays were used in conjunction with cytotoxicitytests. Thus, despite many modifications, the process of producingmonoclonal antibodies relies on antibody binding to the test antigen asan endpoint.

The purified monoclonal antibody is utilized for immunochemical studies.

Polyclonal antibody production and purification utilizing one or moreanimal hosts in a manner well-known in the art can be performed by askilled artisan.

Another objective of the present invention is to provide reagents foruse in diagnostic assays for the detection of the particularly isolateddisease specific marker sequences of the present invention.

In one mode of this embodiment, the marker sequences of the presentinvention may be used as antigens in immunoassays for the detection ofthose individuals suffering from the disease known to be evidenced bysaid marker sequence. Such assays may include but are not limited to:radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), “sandwich”assays, precipitin reactions, gel diffusion immunodiffusion assay,agglutination assay, fluorescent immunoassays, protein A or Gimmunoassays and immunoelectrophoresis assays.

According to the present invention, monoclonal or polyclonal antibodiesproduced against the disease specific marker sequence of the instantinvention are useful in an immunoassay on samples of blood or bloodproducts such as serum, plasma or the like, cerebrospinal fluid or otherbody fluid, e.g. saliva, urine, lymph, and the like, to diagnosepatients with the characteristic disease state linked to said markersequence. The antibodies can be used in any type of immunoassay. Thisincludes both the two-site sandwich assay and the single siteimmunoassay of the non-competitive type, as well as in traditionalcompetitive binding assays.

Particularly preferred, for ease and simplicity of detection, and itsquantitative nature, is the sandwich or double antibody assay of which anumber of variations exist, all of which are contemplated by the presentinvention. For example, in a typical sandwich assay, unlabeled antibodyis immobilized on a solid phase, e.g. microtiter plate, and the sampleto be tested is added. After a certain period of incubation to allowformation of an antibody-antigen complex, a second antibody, labeledwith a reporter molecule capable of inducing a detectable signal, isadded and incubation is continued to allow sufficient time for bindingwith the antigen at a different site, resulting with a formation of acomplex of antibody-antigen-labeled antibody. The presence of theantigen is determined by observation of a signal which may bequantitated by comparison with control samples containing known amountsof antigen.

Antibodies may also be utilized against the disease specific markers, ashaptens, to create an antibody response against the protein to which itbinds, thereby identifying targets for treatment of the disease or asub-class thereof.

Lastly, the markers and associated antibodies provide a tool formonitoring the progress of a patient during a therapeutic treatment, soas to determine the usefulness of a novel therapeutic agent.

All patents and publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

It is to be understood that while a certain form of the invention isillustrated, it is not to be limited to the specific form or arrangementherein described and shown. It will be apparent to those skilled in theart that various changes may be made without departing from the scope ofthe invention and the invention is not to be considered limited to whatis shown and described in the specification and drawings/figures.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objectives and obtain theends and advantages mentioned, as well as those inherent therein. Theoligonucleotides, peptides, polypeptides, biologically relatedcompounds, methods, procedures and techniques described herein arepresently representative of the preferred embodiments, are intended tobe exemplary and are not intended as limitations on the scope. Changestherein and other uses will occur to those skilled in the art which areencompassed within the spirit of the invention and are defined by thescope of the appended claims. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in the art are intended to be within the scope of thefollowing claims.

1. A method for diagnosing Type II diabetes comprising: (a) providing asample; and (b) determining the presence of a biopolymer markerconsisting of SEQ ID NO:4 in said sample; wherein the presence of saidbiopolymer marker consisting of SEQ ID NO:4 is diagnostic for Type IIdiabetes.
 2. The method of claim 1, wherein said sample isan-unfractionated body fluid or a tissue sample.
 3. The method of claim1, wherein said sample is selected from the group consisting of blood,blood products, urine, saliva, cerebrospinal fluid, and lymph.
 4. Themethod of claim 1, wherein said sample is obtained from a human patient.5. A method for screening for efficacy of disease process modulatingagents for Type II diabetes comprising: (a) providing a sample of bodilyfluid containing a biopolymer marker consisting of SEQ ID NO:4; (b)adding a quantity of said agent sufficient to interact with saidbiopolymer marker consisting of SEQ ID NO:4; and (c) determining thepresence of an interaction between said agent and said biopolymer markerconsisting of SEQ ID NO:4; wherein said interaction is determinative ofefficacy of said disease process modulating agent.
 6. A method fordiagnosing Type II diabetes by determining the presence of a biopolymermarker consisting of SEQ ID NO:4 comprising: (a) conducting massspectrometric analysis on a sample obtained from a patient in a mannereffective to maximize analysis of peptide fragments contained therein;(b) comparing a mass spectral profile of said biopolymer markerconsisting of SEQ ID NO:4 to mass spectral profiles of peptides obtainedand analyzed from said sample; and (c) confirming the presence of saidbiopolymer marker consisting of SEQ ID NO:4 in said sample byidentifying a mass spectral profile having an ion peak at about 1353daltons; wherein the presence of said biopolymer marker consisting ofSEQ ID NO:4 is diagnostic for Type II diabetes.
 7. The method of claim6, wherein said sample is an unfractionated body fluid or a tissuesample.
 8. The method of claim 6, wherein said sample is selected fromthe group consisting of blood, blood products, urine, saliva,cerebrospinal fluid, and lymph.
 9. The method of claim 6, wherein saidmass spectrometric analysis is selected from the group consisting ofSurface Enhanced Laser Desorption Ionization (SELDI) mass spectrometry,Maldi Qq TOF, MS/MS, TOF-TOF, ESI-Q-TOF and ION-TRAP.
 10. The method ofclaim 6, wherein said patient is a human.